Provided is a method of preparing amino linker oligonucleotides with a high yield by efficiently removing an amino protecting group from amino linker oligonucleotides protected with the protecting group, and a method of preparing labeled linker oligonucleotides with a high yield from the amino linker oligonucleotides.
In the preparation of labeled oligonucleotides, the following two methods have generally been used to introduce a label such as biotin, fluorescein, thiol, cholesterol, and the like, to 5′ terminus of oligonucleotides; a solid phase labeling technique (phosphoramidite coupling) using an expensive labeling phosphoramidite agents, and a liquid phase labeling technique using labeling agents in the form of N-hydroxy succinimide ester.
Examples of the agents used for the phosphoramidite coupling include fluorescein phosphoramidite, cholesteryl phosphoramidite, 5′-biotin phosphoramidite, thiol-modifier C6 SS, and the like. There is a problem that the agents used for the phosphoramidite coupling are too expensive. Another problem is that the agents once resolved in liquid are so unstable that reaction yields decrease quickly, as the storage time is longer. FIG. 1 briefly illustrates a labeling method using the phosphoramidite coupling, wherein the prices of the labeling agents used are also recited.
Meanwhile, the liquid phase labeling method using agents in the form of N-hydroxy succinimide ester has difficulties in separation and filtration, and thus, there are some limits in using the liquid phase labeling for the preparation of labeled oligonucleotides.
Besides, there are other methods using amino linkers to prepare labeled oligonucleotides. The method for preparing labeled oligonucleotides using amino linkers generally has a problem of low yields compared to when the solid phase phosphoramidite coupling is employed. That is because amino protecting groups are not completely removed from oligonucleotides, unlike peptides, such that the yield of deprotected amino linker oligonucleotides is lowered. A yield for the removal of amino protecting groups is reported to be approximately 50% in the method for preparing labeled oligonucleotides using amino linkers (Zaramella, S., et al, J. Am. Chem. Soc., 2004, 126, 14029-14035).
In this context, there is a growing interest in developing an economic method for amino linker oligonucleotides synthesis by quantitatively removing amino protecting groups and achieving a higher yield than that in the phosphoramidite coupling.